Somatic hypermutation introduces single base changes into rearranged and expressed immunoglobulin variable regions, to increase the affinity of the immune response or the diversity of the immune repertoire. In the past few years, there has been extraordinary progress toward understanding the regulation and mechanism of hypermutation. In particular, the demonstration that DNA breaks are associated with hypermutation has verified our hypothesis that hypermutation proceeds by a break and repair pathway. We will build upon this to identify factors involved in initiating hypermutation and to define the subsequent steps in the mutagenic repair processes that alter immunoglobulin gene sequence. We propose (1) to characterize DNA breaks associated with hypermutation in the chicken B cell line DT40; (2) to identify nuclease activities that cleave DNA in the first step of hypermutation; (3) to ask how and when MSH2 functions in hypermutation; (4) to study the response of mutagenic repair pathways to induction of DNA breaks in hypermutating B cells; and (5) to visualize dynamic interactions of mutagenic repair factors in hypermutating B cells, using fluorescent microscopy. Hypermutation is essential to a normal immune response, but the cleavage and mutagenic repair activities associated with hypermutation can also function outside this pathway, leading to genomic destabilization and tumorigenesis. The proposed experiments will provide us with molecular targets which can be manipulated to enhance the efficacy of immunization and to minimize genomic instability in normal cells and cancer cells.